In the hippocampus, the cAMP dependent protein kinase (PKA) plays critical roles in neurotransmission, cell excitability, and synaptic plasticity.All of these processes require high specificity in PKA phosphorylation. Previous studies have shown that PKA specificity is established by a class of proteins called A-kinase anchoring proteins (AKAPs), which target upstream activators and downstream substrates of PKA to specific subcellular locations. However, AKAPs only bind the regulatory subunit of PKA. Once activated by cAMP, the catalytic subunit of PKA is released from the regulatory subunit and is free to diffuse. A cytosolic protein of comparable size to PKA will equilibrate synaptic compartments, such as dendritic spines, in tens of milliseconds and exit these compartments within a hundreds of milliseconds. As these time scales are significantly shorter than the known time scales of PKA signaling, such diffusion would be expected to break down the PKA specificity established by AKAPs. How does PKA signaling maintain its specificity despite the freely diffusing catalytic subunit? Our preliminary results suggest a novel mechanism for PKA specificity beyond AKAPs: constrained diffusion of the PKA catalytic subunits. To test this hypothesis, and to further investigate PKA dynamics in neurons, we have built a custom two-photon microscope capable of localized photoactivation and two-photon fluorescence lifetime imaging (2pFLIM). The photoactivation approach enables us to measure PKA diffusion in synaptic compartments and 2pFLIM allows us to monitor PKA activity at the resolution of signal spines using PKA sensors based on fluorescence resonance energy transfer (FRET). Taking advantage of these techniques we propose to: 1) identify the molecular components of PKA responsible for its restricted diffusion and their functional significance, and 2) determine the spatiotemporal dynamics of PKA in response to both applied and endogenous stimuli. The goal of the experiments in this application is to incorporate PKA diffusion into a model of PKA specificity in post-synaptic compartments.